Blood cells have a relatively short life span and need to be replenished throughout life. In adults, blood cell formation or hematopoiesis takes place in the bone marrow, but blood-forming stem cells can also be found in peripheral blood. Hematopoietic cells represent a hierarchy of proliferating and differentiating cells. The most abundant are the differentiating cells. These cells have limited or no proliferative capacity and represent the immediate precursors of the specialized end cells that are found in blood. The immediate precursors of the differentiating cells are the progenitor cells. Most of these cells are restricted to differentiate along a single lineage but they may have quite extensive proliferative capacity. Progenitor cells appear morphologically as blast cells and they typically do not have specific features of the hematopoietic lineage to which they are committed. Progenitor cells are derived from stem cells. Stem cells have been historically defined by their ability to self-renew as well as to generate daughter cells of any of the hematopoietic lineages. The presence of stem and progenitor cells may be detected by their ability to produce colony-forming cells in culture. They may also be detected by screening for the CD34 antigen which is a positive marker for early hematopoietic cells including colony forming cells and stem cells.
There is a continued interest in developing stem cell purification techniques. Pure populations of stem cells will facilitate studies of hematopoiesis. Transplantation of hematopoietic cells from peripheral blood and/or bone marrow is also increasingly used in combination with high-dose chemo- and/or radiotherapy for the treatment of a variety of disorders including malignant, non-malignant and genetic disorders. Very few cells in such transplants are capable of long-term hematopoietic reconstitution and thus there is a strong stimulus to develop techniques for purification of hematopoietic stem cells. Furthermore, serious complications and indeed the success of a transplant procedure is to a large degree dependent on the effectiveness of the procedures that are used for the removal of cells in the transplant that pose a risk to the transplant recipient. Such cells include T lymphocytes that are responsible for graft versus host disease (GVHD) in allogeneic grafts and tumour cells in autologous transplants that may cause recurrence of the malignant growth.
A variety of techniques have been described for the removal of either T cells or tumour cells from transplants (See for example Bone Marrow Processing and Purging: A Practical Guide, (ed. A. P. Gee), CRC Press, Boca Raton (1991)). Most of the techniques involve purification of the hematopoietic cells ("positive selection") or the depletion or "purging" of tumour cells ("negative selection") in the cell preparation used for transplantation.
The two most important variables in either positive or negative selection techniques are (1) the efficiency of removal of undesirable cells (either T cells or tumor cells) and (2) the recovery of hematopoietic cells (most readily assessed by measurement of CD34 positive cells before and after the separation). These variables are typically expressed as (1) the logarithm (log) of the depletion and (2) the percentage recovery of the CD34 positive cells. For example, a technique for depleting T cells in a cell suspension that results in a two log depletion of T cells, and a 30% recovery of CD34 positive cells, would provide a cell suspension containing 1% of the T cells and 30% of CD34 positive cells that were present in the cell suspension before the T cell depletion procedure.
High gradient magnetic separation (HGMS) has been used for the removal of magnetically labelled cells from suspensions of bone marrow cells for research use (Bieva et al., Exp. Hematol. (1989) 17: 914; Miltenyi et al., Cytometry (1990) 11: 231; and Kogler et al., Bone Marrow Transplant. (1990) 6: 163 and Thomas et al., J. Hematother. (1993) 2: 297; and clinical use (Yau et al., Exp. Hematol. (1990) 18: 219; Poynton et al., The Lancet (1983) March: 524; and Reading et al., Leukemia Res. (1987) 11: 1067).
HGMS separation involves placing a filter of fine magnetisable wires in a strong magnetic field. High gradient magnetic fields are produced around the wires, allowing the capture of even very weakly magnetic particles upon the magnetisable wires.
There have been several attempts to apply HGMS to the separation and isolation of magnetically labelled CD34 positive cells (i.e. positive selection techniques), although the recoveries and purities achieved have been undesirably low (For example, see Kato, K., and Radbruch, A., Cytometry 14:384, 1993). Typically, attempts have employed an HGMS filter which consists of a random or semi-random array of stainless steel wire wound loosely into a column located in a strong magnetic field (Miltenyi, S. et al., Cytometry 11:231, 1990; Molday, R. S. and Molday, L., FEBS. Lett. 170:232, 1984; Kato, K and Radbruch, A., supra; Kemshead, J. T. in Hematotherapy 1:35, 1992; and Kemshead, J. T. in Bone Marrow Processing and Purging, 293, Gee, A. P. Ed., C. R. C. Press, Inc., Boca Raton, Fla., 1991). The positive selection procedures suffer from many disadvantages including the presence of materials such as antibodies and/or magnetic beads on the CD34 positive cells, and damage to the cells resulting from the removal of these materials.
It has been assumed that pure hematopoietic stem cells can be numerically expanded in the laboratory. Accordingly, investigators typically have not focused on the recovery or yield of hematopoietic stem cells that can be obtained with the available cell purification or cell "purging" methods. However, recent studies with highly purified candidate stem cells from human and murine bone marrow have shown that it may not be possible to achieve such numerical expansion of stem cells derived from adult hematopoietic tissue in vitro (Lansdorp et al., J. Exp. Med. (1993) 178: 787, and Rebel et al., Blood (1994) 83:128). As a result, techniques that optimize the use of the available hematopoietic cells for transplantation are of considerable interest. Unfortunately, all currently available methods for the removal of either T cells or tumour cells from transplants suffer from deficiencies. These include the following: 1) the methods that allow for effective (i.e. &gt;2 log) depletion of T or tumour cells typically recover generally far less than 50% of the normal blood-forming or hematopoietic cells initially present in the cell suspension available for transplantation (i.e. as a result of centrifugation, density separation, wash procedures and other preprocessing required prior to the actual separation process or during the immunological selection procedure itself); and/or 2) the methods that recover &gt;50% of the hematopoietic cells fail to reproducibly achieve effective (&gt;2 log) depletion of undesirable cells.